Antibodies against Frizzled receptor

ABSTRACT

This invention provides monoclonal antibodies that recognize one or more Frizzled receptors. The invention further provides methods of using such monoclonal antibodies as a therapeutic, diagnostic, and prophylactic.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/911,983, filed on Feb. 12, 2016, which is a national stageapplication filed under 35 U.S.C. § 371, of International ApplicationNo. PCT/US2014/051070, filed Aug. 14, 2014, which claims the benefit ofU.S. Provisional Application No. 61/865,668, filed Aug. 14, 2013, thecontents of which are herein incorporated by reference in theirentirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the file named “UTOR-002_C01US SequenceListing_ST25.txt”, which was created on Aug. 1, 2018 and is 83.3 KB insize, are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to the generation of antibodies, e.g.,monoclonal antibodies, that recognize one or more Frizzled receptors,and to methods of using these anti-Frizzled antibodies as therapeutics.

BACKGROUND OF THE INVENTION

Frizzled receptors belong to a class of G protein-coupled receptors.Aberrant Frizzled receptor expression or activity has been implicated invarious disorders. Accordingly, there exists a need for therapies thattarget and inhibit one or more Frizzled receptors.

SUMMARY OF THE INVENTION

The present invention provides high affinity antibodies such asmonoclonal antibodies which recognize a Frizzled receptor or acombination of Frizzled receptors, for example, a human Frizzledreceptor or a combination of human Frizzled receptors. In someembodiments, the antibodies bind to the cysteine rich domain (CRD) of aFrizzled receptor or the CRDs of a combination of Frizzled receptors.These antibodies are capable of modulating, e.g., blocking, inhibiting,reducing, antagonizing, neutralizing or otherwise interfering with oneor more biological activities of a Frizzled receptor or a combination ofFrizzled receptors, for example, binding to a Wnt protein ligand, whichactivates a Wnt signaling pathway.

These antibodies and antigen-binding fragments thereof bind to one ormore Frizzled receptors expressed on the cell surface. For example,these antibodies bind to one or more Frizzled receptors on variouscancer cell lines and inhibit the growth of cancer cells of multipletissue origins. These antibodies also bind to one or more Frizzledreceptors on various cancer cell lines and inhibit the growth of cancerstem cells. Thus, the antibodies and antigen-binding fragments thereofare useful for treating, preventing, delaying the progression of orotherwise ameliorating a symptom of cancer, as well as other diseaseswhere Frizzled receptor expression and/or activity is dysregulated suchas, by way of non-limiting example, bone diseases, including, e.g.,osteoporosis, osteoarthritis (OA), and rheumatoid arthritis (RA). Insome embodiments, the antibodies and antigen-binding fragments thereofare useful for treating a cancer, such as, by way of non-limitingexample, breast cancer, including triple negative breast cancer, lungcancer, colon cancer, ovarian cancer, pancreatic cancer,gastrointestinal (GI) cancer, neuroblastoma, renal cancer, prostatecancer, melanoma, leukemia, and/or Wilm's tumor. In some embodiments,the antibodies and antigen-binding fragments thereof are useful fortreating a cancer that is associated with cancer stem cells. In someembodiments, these anti-Frizzled receptor antibodies and fragments ofthe invention are useful in modulating, e.g., blocking, inhibiting,reducing, antagonizing, neutralizing or otherwise interfering with thesurvival, migration and/or invasion, e.g., metastasis, of a cancer cell.In some embodiments, these anti-Frizzled receptor antibodies andfragments of the invention are useful in modulating, e.g., blocking,inhibiting, reducing, antagonizing, neutralizing or otherwiseinterfering with the survival, migration and/or invasion, e.g.,metastasis, of a breast cancer cell, a lung cancer cell, a colon cancercell and/or an ovarian cancer cell.

Exemplary antibody fragments of the invention include, for example, theFab fragments having the complementarity determining region (CDR)sequences shown in FIG. 1C and encoded by the sequences shown in FIG.1D, where the variable light chain complementarity determining region 1(CDR L1 or VL CDR1) includes the amino acid sequence SVSSA (SEQ ID NO:392) and the variable light chain complementarity determining region 2(CDR L2 or VL CDR2) includes the amino acid sequence SASSLYS (SEQ ID NO:393). Exemplary monoclonal antibodies of the invention include, forexample, antibodies having the light chain and heavy chain sequencesshown in FIGS. 1A and 1B. Exemplary monoclonal antibodies of theinvention include, for example, IgG antibodies having the combination ofcomplementarity determining regions (CDRs) shown in FIG. 1C and encodedby the sequences shown in FIG. 1D, where the CDR L1 includes the aminoacid sequence SVSSA (SEQ ID NO: 392) and the CDR L2 includes the aminoacid sequence SASSLYS (SEQ ID NO: 393).

In some embodiments, the monoclonal antibody is an antibody or anantigen binding fragment thereof that binds to the same Frizzled epitopeas antibodies having the light chain and heavy chain sequences shown inFIGS. 1A and 1B, or the antibody fragment is a fragment that binds tothe same Frizzled epitope as the Fab fragments having the sequencesshown in FIG. 1C and encoded by the sequences shown in FIG. 1. In someembodiments, the antibody or antigen binding fragment thereof inhibitsinteraction between one or more Frizzled receptors and one or more Wntprotein ligands. In some embodiments, the antibody or antigen bindingfragment thereof inhibits Wnt signaling. In some embodiments, theantibody fragment is a fragment of an antibody that binds to the sameFrizzled epitope as antibodies having the light chain and heavy chainsequences shown in FIGS. 1A and 1B. In some embodiments, the antibody orantigen binding fragment thereof inhibits interaction between one ormore Frizzled receptors and one or more Wnt protein ligands. In someembodiments, the antibody or antigen binding fragment thereof inhibitsWnt signaling. These antibodies are collectively referred to herein as“anti-Frizzled receptor antibodies,” and these fragments arecollectively referred to herein as “anti-Frizzled receptor antibodyfragments.” In some embodiments, the antibody or immunologically activefragment thereof that binds one or more Frizzled receptors is amonoclonal antibody, domain antibody, single chain, Fab fragment, aF(ab′)₂ fragment, a scFv, a scab, a dAb, a single domain heavy chainantibody, and a single domain light chain antibody. In some embodiments,such an antibody or immunologically active fragment thereof that bindsone or more Frizzled receptors is a mouse, chimeric, humanized or fullyhuman monoclonal antibody. These antibodies show specificity for one ormore Frizzled receptors, preferably one or more human Frizzledreceptors, and they have been shown to modulate, e.g., block, inhibit,reduce, antagonize, neutralize or otherwise interfere with at least onebiological activity of one or more Frizzled receptors, preferably, oneor more human Frizzled receptors.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a heavy chain region having the amino acid sequence ofSEQ ID NO: 4. In some embodiments, the antibodies and antigen-bindingfragments thereof contain a light chain region having the amino acidsequence of SEQ ID NO: 2. In some embodiments, the antibodies andantigen-binding fragments thereof contain a heavy chain region havingthe amino acid sequence of SEQ ID NO: 4 and a light chain region havingthe amino acid sequence of SEQ ID NO: 2.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a heavy chain region having an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 4. In some embodiments, theantibodies and antigen-binding fragments thereof contain a light chainregion having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence ofSEQ ID NO: 2. In some embodiments, the antibodies and antigen-bindingfragments thereof contain a heavy chain region having an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 4, and a lightchain region having an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequenceof SEQ ID NO: 2.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a heavy chain region that is encoded by the nucleic acidsequence of SEQ ID NO: 3. In some embodiments, the antibodies andantigen-binding fragments thereof contain a light chain region that isencoded by the nucleic acid sequence of SEQ ID NO: 1. In someembodiments, the antibodies and antigen-binding fragments thereofcontain a heavy chain region that is encoded by the nucleic acidsequence of SEQ ID NO: 3 and a light chain region that is encoded by thenucleic acid sequence of SEQ ID NO: 1.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a heavy chain region that is encoded by a nucleic acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%,99% or more identical to the nucleic acid sequence of SEQ ID NO: 3. Insome embodiments, the antibodies and antigen-binding fragments thereofcontain a light chain region that is encoded by a nucleic acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or moreidentical to the nucleic acid sequence of SEQ ID NO: 1. In someembodiments, the antibodies and antigen-binding fragments thereofcontain a heavy chain region that is encoded by a nucleic acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or moreidentical to the nucleic acid sequence of SEQ ID NO: 3, and a lightchain region that is encoded by a nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to thenucleic acid sequence of SEQ ID NO: 1.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a heavy chain region with three heavy chain CDRs wherethe variable heavy chain (VH) complementarity determining region 1 (CDRH1) includes an amino acid sequence selected from the group consistingof those shown in FIG. 1C; the VH complementarity determining region 2(CDR H2) includes an amino sequence selected from the group consistingof those shown in FIG. 1C; and the VH complementarity determining region3 (CDR H3) includes an amino acid sequence selected from the groupconsisting of those shown in FIG. 1C.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a heavy chain region with three heavy chain CDRs wherethe CDR H1 includes an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98%, 99% or more identical to an amino sequenceselected from the group consisting of those shown in FIG. 1C; the CDR H2includes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97% 98%, 99% or more identical to an amino sequence selected fromthe group consisting of those shown in FIG. 1C; and the CDR H3 includesan amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%98%, 99% or more identical to an amino sequence selected from the groupconsisting of those shown in FIG. 1C.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a light chain region with three light chain CDRs wherethe variable light chain (VL) CDR1 (CDR L1) includes the sequence of SEQID NO: 392; the CDR L2 includes the amino acid sequence of SEQ ID NO:393; and the CDR L3 includes an amino acid sequence selected from thegroup consisting of those shown in FIG. 1C.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a light chain region with three light chain CDRs wherethe CDR L1 includes an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98%, 99% or more identical to the sequence of SEQ IDNO: 392; the CDR L2 includes an amino acid sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the aminoacid sequence of SEQ ID NO: 393; and the CDR L3 includes an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% ormore identical to an amino sequence selected from the group consistingof those shown in FIG. 1C.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a heavy chain region with three heavy chain CDRs andthree light chain CDRs where the CDR H1 includes an amino acid sequenceselected from the group consisting of those shown in FIG. 1C; the CDR H2includes an amino sequence selected from the group consisting of thoseshown in FIG. 1C; the CDR H3 includes an amino acid sequence selectedfrom the group consisting of those shown in FIG. 1C; the CDR L1 includesthe sequence of SEQ ID NO: 392; the CDR L2 includes the amino acidsequence of SEQ ID NO: 393; and the CDR L3 includes an amino acidsequence selected from the group consisting of those shown in FIG. 1C.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a heavy chain region with three heavy chain CDRs andthree light chain CDRs where the CDR H1 includes an amino acid sequenceat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or moreidentical to an amino sequence selected from the group consisting ofthose shown in FIG. 1C; the CDR H2 includes an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identicalto an amino sequence selected from the group consisting of those shownin FIG. 1C; the CDR H3 includes an amino acid sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to an aminosequence selected from the group consisting of those shown in FIG. 1C;the CDR L1 includes an amino acid sequence at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98%, 99% or more identical to the sequence of SEQ IDNO: 392; the CDR L2 includes an amino acid sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the aminoacid sequence of SEQ ID NO: 393; and the CDR L3 includes an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% ormore identical to an amino sequence selected from the group consistingof those shown in FIG. 1C.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a heavy chain region having the amino acid sequence ofSEQ ID NO: 4, where one or more of the heavy chain CDR sequences isreplaced with the corresponding CDR H1, CDR H2 and/or CDR H3 sequenceselected from those listed in FIG. 1C and encoded by the sequences shownin FIG. 1D. In some embodiments, the antibodies and antigen-bindingfragments thereof contain a light chain region having the amino acidsequence of SEQ ID NO: 2, where the CDR L3 is replaced with a CDR L3sequence selected from those listed in FIG. 1C and encoded by thesequences shown in FIG. 1D. In some embodiments, the antibodies andantigen-binding fragments thereof contain a heavy chain region havingthe amino acid sequence of SEQ ID NO: 4, where one or more of the heavychain CDR sequences is replaced with the corresponding CDR H1, CDR H2and/or CDR H3 sequence selected from those listed in FIG. 1C and encodedby the sequences shown in FIG. 1D, and a light chain region having theamino acid sequence of SEQ ID NO: 2, where the CDR L3 is replaced with aCDR L3 sequence selected from those listed in FIG. 1C and encoded by thesequences shown in FIG. 1D.

In some embodiments, the antibodies and antigen-binding fragmentsthereof contain a heavy chain region having an amino acid sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 4, where one or more of theheavy chain CDR sequences is replaced with the corresponding CDR H1, CDRH2 and/or CDR H3 sequence selected from those listed in FIG. 1C andencoded by the sequences shown in FIG. 1D. In some embodiments, theantibodies and antigen-binding fragments thereof contain a light chainregion having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence ofSEQ ID NO: 2, where the CDR L3 is replaced with a CDR L3 sequenceselected from those listed in FIG. 1C and encoded by the sequences shownin FIG. 1D. In some embodiments, the antibodies and antigen-bindingfragments thereof contain a heavy chain region having an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 4, where one ormore of the heavy chain CDR sequences is replaced with the correspondingCDR H1, CDR H2 and/or CDR H3 sequence selected from those listed in FIG.1C and encoded by the sequences shown in FIG. 1D, and a light chainregion having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence ofSEQ ID NO: 2, where the CDR L3 is replaced with a CDR L3 sequenceselected from those listed in FIG. 1C and encoded by the sequences shownin FIG. 1D.

In some embodiments, an exemplary antibody or antigen-binding fragmentsthereof contain a CDR L1 that includes the amino acid sequence SVSSA(SEQ ID NO: 392), a CDR L2 that includes the amino acid sequence SASSLYS(SEQ ID NO: 393), a CDR L3 region that includes the amino acid sequenceWAYGPF (SEQ ID NO: 53), a CDR H1 region that includes the amino acidsequence IYYYSM (SEQ ID NO: 54), a CDR H2 region that includes the aminoacid sequence SIYSSYSYTS (SEQ ID NO: 19), and a CDR H3 region thatincludes the amino acid sequence SSPGADYGL (SEQ ID NO: 55). Thisantibody is referred to herein as H10 or mAb #111, which are usedinterchangeably throughout the disclosure.

In some embodiments, an exemplary antibody or antigen binding fragmentsthereof contain a CDR L1 that includes the amino acid sequence SVSSA(SEQ ID NO: 392), a CDR L2 that includes the amino acid sequence SASSLYS(SEQ ID NO: 393), a CDR L3 region that includes the amino acid sequenceGVYLF (SEQ ID NO: 112), a CDR H1 region that includes the amino acidsequence IYSSSI (SEQ ID NO: 113), a CDR H2 region that includes theamino acid sequence SIYSSYGSTS (SEQ ID NO: 114), and a CDR H3 regionthat includes the amino acid sequence YHYPFGHAL (SEQ ID NO: 115). Thisantibody is referred to herein as G2 or mAb #140, which are usedinterchangeably throughout the disclosure.

In some embodiments, an exemplary antibody or antigen binding fragmentsthereof contain a CDR L1 that includes the amino acid sequence SVSSA(SEQ ID NO: 392), a CDR L2 that includes the amino acid sequence SASSLYS(SEQ ID NO: 393), a CDR L3 region that includes the amino acid sequenceYYHPI (SEQ ID NO: 159), a CDR H1 region that includes the amino acidsequence ISSYYI (SEQ ID NO: 150), a CDR H2 region that includes theamino acid sequence SIYPYYSSTY (SEQ ID NO: 160), and a CDR H3 regionthat includes the amino acid sequence VWYGAM (SEQ ID NO: 161). Thisantibody is referred to herein as A1 or mAb #105, which are usedinterchangeably throughout the disclosure.

In some embodiments, an exemplary antibody or antigen binding fragmentsthereof contain a CDR L1 that includes the amino acid sequence SVSSA(SEQ ID NO: 392), a CDR L2 that includes the amino acid sequence SASSLYS(SEQ ID NO: 393), a CDR L3 region that includes the amino acid sequenceSSYSLI (SEQ ID NO: 71), a CDR H1 region that includes the amino acidsequence LSYYSM (SEQ ID NO: 93), a CDR H2 region that includes the aminoacid sequence SIYPSYGYTY (SEQ ID NO: 84), and a CDR H3 region thatincludes the amino acid sequence PSPGSYHGM (SEQ ID NO: 94). Thisantibody is referred to herein as E4 or mAb #107, which are usedinterchangeably throughout the disclosure.

In some embodiments, an exemplary antibody or antigen binding fragmentsthereof contain a CDR L1 that includes the amino acid sequence SVSSA(SEQ ID NO: 392), a CDR L2 that includes the amino acid sequence SASSLYS(SEQ ID NO: 393), a CDR L3 region that includes the amino acid sequenceYWYGVAPI (SEQ ID NO: 132), a CDR H1 region that includes the amino acidsequence ISSSYI (SEQ ID NO: 133), a CDR H2 region that includes theamino acid sequence YIYSSYGSTY (SEQ ID NO: 134), and a CDR H3 regionthat includes the amino acid sequence ASWYAL (SEQ ID NO: 135). Thisantibody is referred to herein as H1 or mAb #112, which are usedinterchangeably throughout the disclosure.

The present invention also provides methods of treating, preventing,delaying the progression of, or otherwise ameliorating a symptom of oneor more pathologies associated with aberrant Frizzled receptor activity,aberrant Frizzled receptor expression and/or aberrant Wnt signaling byadministering an anti-Frizzled monoclonal antibody of the invention orimmunologically active fragment thereof (e.g., antigen-binding fragment)to a subject in which such treatment or amelioration is desired. Thesubject to be treated is, e.g., human. The monoclonal antibody isadministered in an amount sufficient to treat, prevent or alleviate asymptom associated with the pathology. The amount of monoclonal antibodysufficient to treat or prevent the pathology in the subject is, forexample, an amount that is sufficient to inhibit, reduce or otherwiseantagonize Frizzled receptor binding to a Wnt protein ligand. The amountof monoclonal antibody sufficient to treat or prevent the pathology inthe subject is, for example, an amount that is sufficient to inhibit,reduce or otherwise antagonize Wnt signaling.

Pathologies treated and/or prevented using the anti-Frizzled receptorantibodies and anti-Frizzled receptor antibody fragments of theinvention include, for example, cancer and/or bone diseases.

Pharmaceutical compositions according to the invention can include anantibody or antibody fragment of the invention and a carrier. Thesepharmaceutical compositions can be included in kits, such as, forexample, diagnostic kits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a series of illustrations depicting the heavy chainand light chain sequences of Fab H3. As an example, the amino acid andnucleotide sequences of CDRs and backbone are shown. The CDRs areunderlined. The amino acid residues of CDRs listed are the followingpositions inclusive as designed by the IMGT nomenclature: CDR L3:107-116; CDR H1: 30-39; CDR H2: 55-66, CDR H3: 107-115.

FIGS. 1C and 1D are a series of tables depicting the sequences of theCDRs (L3, H1, H2, and H3). Unique Fab sequences from the selections areshown. Clonal phage were amplified in 96-well culture boxes for ELISAscreening. Ninety-five clones from round 2 and ninety-five clones fromround 3 were screened for ELISA reactivity to the FZD7-CDR-Fc domain anda control Fc protein. A total of 57/95 phage clones from Round 3 and59/95 phage clones from Round 2 showed signal to background ratiosgreater than three. Phage clones were sequenced to determine the CDR L3,H1, H2, and H3 compositions. Amino acid residues listed are thefollowing positions inclusive as designated by the IMGT nomenclature:CDR L3: 107-116; CDR H1: 30-39; CDR H2: 55-66, CDR H3: 107-115.

FIG. 2A is a table depicting the purification summary of Fabs. UniqueFabs isolated from library screens were subcloned into an IPTG inducibleexpression vector and expressed in 25 ml small scale cultures. Lysatesof overnight culture pellets were batch purified on Protein A beads andsubjected to two sequential elutions. Yields of each elution and thetotal Fab yields are indicated.

FIG. 2B is a table depicting the binding of purified Fabs to purifiedFZD7-Fc measured by ELISA. Fabs purified from 25 ml bacterial cultureswere assayed for binding to FZD7-Fc fusion protein (R&D systems) and Fcprotein. ELISA plates (384 well) were coated with 2 ug/ml of protein inPBS overnight at four degrees. Wells were blocked with 0.5% BSA/PBS for1 hour at room temperature, washed three times with 0.05% Tween20/PBS(wash buffer), and then primary dilutions of Fabs in 0.5% BSA/0.05%Tween20/PBS (dilution buffer) were added at indicated concentrations.Fabs were incubated for 1 hour at room temperature, wells were washedsix times, and anti-FLAG-HRP (Sigma) was added at 1:5000 in dilutionbuffer. Secondary antibody was incubated for 45 minutes at roomtemperature, and wells were washed six times and developed with TMBsubstrate with an acid stop. The absorbance at 450 nm was read. n.t.=nottested.

FIG. 3 is a table depicting the binding of the purified Fabs to cellsexpressing FZD7 (flow cytometry). Small scale Fabs were assayed forbinding to full-length FZD7 receptor to an over-expression line in MDAMB 231 cells. In parallel, Fabs were assayed for binding to endogenousreceptor expressed on the MDA MB 231 cells. Cells were harvested usingan EDTA solution, blocked in 2% FBS/PBS (stain buffer), and stained with200 nM Fab for 30 min on ice. Cells were washed two times with stainbuffer and incubated with anti-F(ab′)2-APC secondary antibody (JacksonImmuno) at 1:1000 for 15 minutes on ice. All antibodies were diluted instain buffer. Cells were washed three times with stain buffer and thenfixed with 1% paraformaldehye for analysis by flow cytometry. The livecell population was gated based on forward and side scatter profiles,and then a positive fluorescence gate was set against a negative controlFab that did not bind to the cells. The percent of the cell populationwithin the positive gates is indicated.

FIG. 4A is a table depicting the binding specificity of Fabs displayedon phage determined by ELISA. Phage-Fab clones were assayed forcross-binding by ELISA to FZD CDR-Fc fusion proteins (R&D systems).ELISA plates (384 well) were coated with 2 ug/ml of protein in PBS,overnight at four degrees, and binding phage were detected using ananti-M13-HRP secondary antibody. ELISAs were developed with TMBsubstrate with an acid stop. Positive binding results (OD450 abovebackground signal to an Fc control protein) is indicated (+).

FIG. 4B is a table depicting the binding specificity of Fabs determinedby Immuno-staining. Fabs were evaluated for cross-binding byimmunofluorescence on CHO lines stably expressing GPI-linked CRDdomains. +, binding detected; −, binding not detected.

FIG. 5 is a table depicting the epitope binning of the FZD7 Fab clones(competitive ELISA assay). Fzd7-Fc was coated at 2 ug/ml (384 wellplates) in PBS overnight at 4 degrees. Wells were blocked with 0.5%BSA/PBS for one hour at room temperature, washed three times with 0.05%Tween20/PBS, and then indicated Fabs were diluted to 1 uM in 0.5%BSA/0.05% Tween20/PBS (dilution buffer) and added to the wells. Fabswere incubated for one hour at room temperature and then 10 ul ofindicated phage was added to the well. The added phage were from PEGprecipitation of a 30 ml overnight culture, and were diluted in dilutionbuffer to a concentration previously determined to give an ELISA signalwithin the linear range. Samples were incubated 20 min at roomtemperature and then wells were washed six times. Anti-M13-HRP (1:5000in dilution buffer) was added for 45 minutes at room temperature, andwells were washed six times and developed with TMB substrate with anacid stop. The absorbance at 450 nm was read. The percent binding ofeach phage clone in the presence of the Fabs was determined by dividingthe A450 signal of sample wells by the A450 signal of wells containing anegative control Fab that does not bind the Fzd7-Fc protein.

FIGS. 6A-6J is a table and a series of graphs depicting the bindingaffinity of the anti-FZD7 Fabs measured by Surface Plasmon Resonance(SPR). The affinity parameters are summarized in the table (FIG. 6A).FIGS. 6B-6J show the histograms of the SPR measurements.

FIG. 7 is a table depicting the binding affinity of the anti-FZD7antibodies to additional FZDs measured by SPR. Binding affinities of theFab panel to Fzd CDR-Fc fusion proteins (R&D systems) were assessed bySPR. Human domains were used for Fzd domains 1, 4, 5, 7, and 8. In thecase of Fzd2, a mouse domain showing high sequence identity with humanwas used. ‘x’ denotes cases in which positive binding to the antigen wasnot observed by SPR.

FIG. 8 is a graph depicting the inhibition of Wnt5a binding by anti-FZD7Fabs. Wnt 5a (R&D systems) was biotinylated using a commercial kit(Thermo 21329 EZ-link NHS-PEG4-Biotin) and excess biotin was removed bybuffer exchange using a 3000MWCO Amicon filter. Fzd7-Fc was diluted in1% BSA/PBS (dilution buffer) and incubated with desired Fab or buffersamples for 1 hour at room temperature in 96-well TC plates pre-blockedwith 1% BSA. Control wells with Fc protein were also included.Biotinylated wnt5a was added to the wells and plates were incubated foran additional hour. Control wells in which buffer alone was added inlieu of biotinylated wnt5a were also included. Biotinylated Wnt5a wasadded at a final concentration of 150 ng/ul, Fab proteins were at afinal concentration of 0.5 μM, and Fzd7-Fc was at a final concentrationpreviously determined to give absorbance readings within the linearrange. Fabs BB9 and SR07d were included as negative control samplesgiven their specificity for other protein antigens. Samples weretransferred to pre-blocked streptavidin coated plates (R&D systems) andallowed to capture for 1 hour at room temperature. Wells were washedfour times with 0.05% Tween20/PBS and then anti-Fc-HRP (1:5000 indilution buffer, Jackson Immuno) was added to the wells for 45 min atroom temperature. Wells were washed four times and developed with TMBreagent with an acid stop. The absorbance at 450 nm was read and thepercent binding was calculated as the A450 of the desired Fab welldivided by the A450 of the buffer alone well, multiplied by 100. Errorbars represent three independent ELISA experiments.

FIG. 9 is a graph depicting the effect of anti-FZD7 Fabs on theWnt3a-induced transcriptional activity. A TOP-FLASH receptor system(TCF/LEF binding sites are linked to a luciferase reporter gene) wasstably introduced into MDA MB 231 cells. Fabs at a final concentrationof 400 nM were incubated for 15 hours with Wnt3a conditioned media andappropriate control wells. Wells were lysed and luciferase signals wereread. Error bars represent n=3.

FIG. 10 is a series of graphs depicting the binding of anti-FZD7 IgGs toFZD7 ECD expressed on cell surface. Anti-Fzd7 IgGs were stained by flowcytometry at 25 nM on indicated cell-lines. IgGs were detected using ananti-F(ab)2-FITC labeled secondary antibody (Jackson Immuno), fixed withPFA, and then data was acquired on a BD facscalibur.

FIG. 11 is a series of graphs depicting the effect of anti-FZD7 IgGs onWnt3a-induced transcription. Dose-dependent inhibition curves are shown.The IC₅₀ values are indicated. For IgG G6, only single dose inhibitionwas done. At 200 nM, IgG G6 shows >70%.

FIG. 12A-12E are a series of graphs and tables depicting a dosedependent decrease in the proliferation of pancreatic cancer cell linesfollowing 5 days of exposure to anti-FZD7 monoclonal antibodies. FIG.12A depicts a dose dependent decrease in the proliferation of HPAFIIcells following 5 days exposure to either mAb H10 (also referred toherein as mAb #111) or G2 (also referred to herein as mAb #140).Following exposure to mAb H10 at 10 μg/ml or 50 μg/ml, there was adecrease in proliferation of 24.4% and 38.0%, respectively; followingexposure to mAb G2 at 10 μg/ml or 50 μg/ml there was a decrease inproliferation of 30.4% and 48.0%, respectively. FIG. 12B depicts a dosedependent decrease in the proliferation of IMMPC2 cells following 5 daysof exposure of either mAb H10 or G2. Following exposure to mAb H10 at 10μg/ml or 50 μg/ml, there was a decrease in proliferation of 14.1% and16.2%, respectively; following exposure to mAb G2 at 10 μg/ml or 50μg/ml, there was a decrease in proliferation of 15.7% and 17.9%respectively. FIG. 12C depicts a dose dependent decrease in theproliferation of PANC08.13 cells, following 5 days of exposure of eitherto either mAb H10 or G2. Following exposure to mAb H10 at 10 μg/ml or 50μg/ml, there was a decrease in proliferation of 0.16% and 7.7%,respectively; following exposure to mAb G2 at 10 μg/ml or 50 μg/ml therewas a decrease in proliferation of 4.7% and 7.7%, respectively. FIG. 12Ddepicts a dose dependent decrease in proliferation of ASPC-1 cellsfollowing 5 days of exposure to either mAb H10 or G2. Following exposureto mAb H10 at 10 μg/ml or 50 μg/ml, there was a decrease inproliferation of 37.5% or 41.9%, respectively; following exposure to mAbG2 at 10 μg/ml or 50 μg/ml there was a decrease in proliferation of42.3% and 45.8%, respectively. FIG. 12E depicts a dose dependentdecrease in the proliferation of Capan-2 cells, following 5 days ofexposure to anti-FZD7 mAbs A1 (also referred to herein as mAb #105), E4(also referred to herein as mAb #107), H10 (mAb #111), H1 (also referredto herein as mAb #112), and G2 (mAb #140). The levels of reduction inproliferation were comparable with those found after exposure to the18R5 antibody. Collectively, exposure of the anti-FZD7 mAbs at either 10μg/ml or 50 μg/ml resulted in a decrease in the proliferation of theCapan-2 cells by greater than 35%.

FIG. 13A-13B are a series of graphs and tables depicting a dosedependent response in the proliferation of the cancer cell line APSC-1,following 5 days of incubation with anti-FZD7 antibodies, A1 (mAb #105),E4 (mAb #107), H10 (mAb #111), H1 (mAb #112), G2 (mAb #140) or 18R5.FIG. 13A depicts the dose dependent reduction of proliferation of ASPC-1cells following a 5 day incubation with mAbs A1 (mAb #105), E4 (mAb#107) or H10 (mAb #111) at a concentration of 1 μg/ml, 2 μg/ml, 5 μg/ml,10 μg/ml, or 50 μg/ml. FIG. 13 B depicts a does dependent reduction ofproliferation of ASPC-1 cells following a 5 day incubation with mAbs H1(mAb #112), G2 (mAb #140) or 18R5 at a concentration of 1 μg/ml, 2μg/ml, 5 μg/ml, 10 μg/ml, or 50 μg/ml.

FIG. 14 is a graph and a table depicting in vivo tumor growth inhibitionfollowing administration of anti-FZD7 mAbs to a mouse xenograft tumormodel. Three million AsPC1 cells were transplanted into the flank ofC.B-17 SCID mice, followed by treatment with 20 mg/kg anti-FZD7 mAbstarting on day 6 administered twice per week. All tested anti-FZD7antibodies (i.e. A01 (mAb #105), E04 (mAb #107), H10 (mAb #111), H01(mAb #112), G02 (mAb #140)) demonstrated anti-tumor activity with tumorgrowth inhibition (TGI) ranging from 25%-50%.

FIG. 15 is a graph and a table depicting in vivo dose-dependent tumorgrowth inhibition following administration of anti-FZD7 antibodies, H10(mAb #111) and H01 (mAb #112). Three million AsPC1 cells weretransplanted into the flank of C.B-17 SCID mice, followed by treatmentstarting on day 6 post-transplant with either H10 (mAb #111) or H01(mAb#112) at a concentration of 5 mg/kg, 20 mg/kg, or 50 mg/kg administeredtwice per week. Dose-dependent tumor growth inhibitory activities wereobserved for both H10 (mAb #111) and H01 (mAb #112), with a reduction intumor volume ranging from 36.3-65.8% for H10 (mAb #111) and 35.4%-44.7%for mAb H01 (mAb #112), respectively.

FIG. 16 is a graph depicting dose-dependent tumor growth inhibitionfollowing administration of anti-FZD7 antibody H10 (mAb #111) in aCapan-2 xenograft mouse model. Four million Capan-2 cells weretransplanted into the flank of C.B-17 SCID mice, followed by treatmenton day 6 post-transplant with H10 (mAb #112) at concentrations of either5 mg/kg or 20 mg/kg administered twice per week, for a total 10 doses.Administration of 20 mg/kg resulted in the reduction of tumor volume by52%.

FIG. 17 is a graph depicting dose-dependent tumor growth inhibitionfollowing administration of anti-FZD7 antibody H10 (mAb #111) in aHPAFII xenograft mouse model. Three million HPAFII cells weretransplanted into the flank of C.B-17 SCID mice, followed by twiceweekly treatment on day 6 post-transplant with H10 (mAb #111) for 4.5weeks at a concentration of either 5 mg/kg or 20 mg/kg administered fora total of 8 doses. Administration of 20 mg/kg resulted in the reductionof tumor volume by 94%.

DETAILED DESCRIPTION

The present invention provides high affinity antibodies such asmonoclonal antibodies which recognize one or more Frizzled receptors.Frizzled receptors are an important class of G protein-coupled receptorsthat have been implicated in various biological processes, includingdevelopment, cell proliferation, differentiation, survival and migrationas well as in numerous pathological conditions such as cancers. Theantibodies provided herein bind to a Frizzled receptor or to acombination of Frizzled receptors, block ligand Wnt binding and modulateFrizzled receptor-mediated signaling. Therefore, these antibodies havetherapeutic potential for treating cancer and other diseases whereFrizzled receptors are dysregulated.

Frizzled receptors are involved in many important biological processessuch as development, cell proliferation, survival, migration and stemcell maintenance. Abnormal expression and signaling of these receptorsand their ligands, Wnt proteins, have been associated with numerouscancers, including colon, lung, breast and ovarian cancers. Frequently,multiple Wnt ligands and/or Frizzled receptors are up-regulated and leadto aberrant signaling that drives tumorigenesis. Therefore, inhibitionof multiple Frizzled receptors can be used to achieve increasedanti-cancer efficacy. In addition, Frizzled receptors have also beenimplicated in cancer stem cells, a small population of cancer cells thatare thought to be responsible for drug resistance, tumor relapse andmetastasis. Thus, antagonistic antibodies against Frizzled receptors maybe used to target cancer stem cells and to treat various type of cancer.For example, the antagonistic antibodies against FZD7 provided hereinare useful to treat cancers that express FZD7 and depend on FZD7. Sincethese antibodies also bind to additional FZD receptors, these antibodiesmay be used to treat cancers that express and depend on other Frizzledreceptors.

The Wnt signaling pathways are a group of signal transduction pathwaysmade of proteins that pass signals from outside of a cell through cellsurface receptors to the inside of the cell. Three Wnt signalingpathways have been characterized: the canonical Wnt pathway, thenoncanonical planar cell polarity pathway, and the noncanonicalWnt/calcium pathway. All three Wnt signaling pathways are activated bythe binding of a Wnt-protein ligand to a Frizzled receptor, which passesthe biological signal to the protein Disheveled inside the cell. Thecanonical Wnt pathway leads to regulation of gene transcription, thenoncanonical planar cell polarity pathway regulates the cytoskeletonthat is responsible for the shape of the cell, and the noncanonicalWnt/calcium pathway regulates calcium inside the cell.

The antibodies of the invention modulate the interaction between one ormore Frizzled receptors and a Wnt protein ligand. Frizzled receptorsinclude a Frizzled receptor selected from Frizzled-1 (FZD1), Frizzled-2(FZD2), Frizzled-3 (FZD3), Frizzled-4 (FZD4), Frizzled-5 (FZD5),Frizzled-6 (FZD6), Frizzled-8 (FZD8), Frizzled-9 (FZD9) and Frizzled-10(FZD10). Wnt protein ligands include a human Wnt protein such as, forexample, a human Wnt protein selected from Wnt1, Wnt2, Wnt2B, Wnt3,Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A,Wnt9B, Wnt10A, Wnt10B, Wnt11, and Wnt16. The anti-Frizzled antibodiesinhibit or otherwise antagonize binding to a Wnt protein ligand andmodulate activation of a Wnt signaling pathway. For example, theanti-Frizzled antibodies inhibit or otherwise antagonize binding to aWnt protein ligand and modulate activation of and/or signaling via thecanonical Wnt pathway, the noncanonical planar cell polarity pathway,and/or the noncanonical Wnt/calcium pathway.

In some embodiments, the antibodies bind the cysteine-rich domain (CRD)of FZD7. In some embodiments, the antibodies bind one or more Frizzledreceptors selected from Frizzled-1 (FZD1), Frizzled-2 (FZD2), Frizzled-3(FZD3), Frizzled-4 (FZD4), Frizzled-5 (FZD5), Frizzled-6 (FZD6),Frizzled-7 (FZD7), Frizzled-8 (FZD8), Frizzled-9 (FZD9) and Frizzled-10(FZD10). In some embodiments the antibodies bind more than one Frizzledreceptor selected from FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8,FZD9 and FZD10. For example, in some embodiments, the antibodies bind atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, or at least 9 or more Frizzled receptors.

In some embodiments, the antibodies bind the cysteine-rich domain (CRD)of a Frizzled receptor. In some embodiments, the antibodies bind thecysteine-rich domain (CRD) of one or more Frizzled receptors selectedfrom FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10. Insome embodiments, the antibodies bind the cysteine-rich domain (CRD) ofmore than one Frizzled receptor selected from FZD1, FZD2, FZD3, FZD4,FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10. For example, in someembodiments, the antibodies bind the CRD domains of at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, or atleast 9 or more Frizzled receptors. These antibodies are capable ofmodulating, e.g., blocking, inhibiting, reducing, antagonizing,neutralizing or otherwise interfering with one or more biologicalactivities of one or more Frizzled receptors.

In some embodiments, the antibodies bind Frizzled-7 receptor, alsoreferred to herein as Frizzled-7 and/or FZD7. In some embodiments, theantibodies bind human FZD7. In some embodiments, the antibodies bindFZD7 in combination with one or more Frizzled receptors selected fromFZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD8, FZD9 and FZD10. In someembodiments, the antibodies bind human FZD7 in combination with one ormore human Frizzled receptors selected from human FZD1, human FZD2,human FZD3, human FZD4, human FZD5, human FZD6, human FZD8, human FZD9and human FZD10.

The antibodies of the present invention bind to an epitope on one ormore Frizzled receptors, e.g., an epitope on human FZD7, with anequilibrium binding constant (K_(d)) of ≤1 μM, e.g., ≤100 nM, preferably≤10 nM, and more preferably ≤1 nM. For example, the anti-Frizzledreceptor antibodies and fragments provided herein exhibit a K_(d) in therange shown in FIG. 6 and/or FIG. 7.

The anti-Frizzled receptor antibodies and fragments of the inventionserve to modulate, block, inhibit, reduce, antagonize, neutralize orotherwise interfere with at least one functional activity of one or moreFrizzled receptors. For example, the anti-Frizzled receptor antibodiesand/or anti-Frizzled receptor antibody fragments completely or partiallyinhibit a Frizzled functional activity by partially or completelymodulating, blocking, inhibiting, reducing antagonizing, neutralizing,or otherwise interfering with the binding of one or more Frizzledreceptors to Wnt protein ligand. For example, the anti-Frizzled receptorantibodies and/or anti-Frizzled receptor antibody fragments completelyor partially inhibit Frizzled functional activity by partially orcompletely modulating, blocking, inhibiting, reducing antagonizing,neutralizing, or otherwise interfering with the activation of one ormore Frizzled receptors.

Anti-Frizzled receptor antibodies and/or anti-Frizzled receptor antibodyfragments are considered to completely block at least one functionalactivity of one or more Frizzled receptors when the level of thefunctional activity in the presence of the anti-Frizzled receptorantibody and/or anti-Frizzled receptor antibody fragment is decreased byat least 95%, e.g., by 96%, 97%, 98%, 99% or 100% as compared to thelevel of the functional activity in the absence of interaction, e.g.,binding, with the anti-Frizzled receptor antibody and/or anti-Frizzledreceptor antibody fragment. Anti-Frizzled receptor antibodies and/oranti-Frizzled receptor antibody fragments are considered to partiallyblock at least one functional activity of one or more Frizzled receptorswhen the level of the functional activity in the presence of theanti-Frizzled receptor antibody and/or anti-Frizzled receptor antibodyfragment is decreased by at least 50%, e.g., 55%, 60%, 75%, 80%, 85% or90% as compared to the level of the functional activity in the absenceof interaction, e.g., binding, with the anti-Frizzled receptor antibodyand/or anti-Frizzled receptor antibody fragment.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” “or directed against” is meant that the antibodyreacts with one or more antigenic determinants of the desired antigenand does not react with other polypeptides or binds at much loweraffinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain,F_(ab), F_(ab′) and F_((ab′)2) fragments, scFvs, and an F_(ab)expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or fragment thereof, ora T-cell receptor. The term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. An antibody is said tospecifically bind an antigen when the dissociation constant is ≤1 μM;e.g., ≤100 nM, preferably ≤10 nM and more preferably ≤1 nM.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to one or more Frizzled receptors, when theequilibrium binding constant (K_(d)) is ≤1 μM, preferably ≤100 nM, morepreferably ≤10 nM, and most preferably ≤100 μM to about 1 μM, asmeasured by assays such as radioligand binding assays or similar assaysknown to those skilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the invention include the nucleic acid molecule encodingthe heavy chain immunoglobulin molecule presented in SEQ ID NO: 4, e.g.,the nucleic acid sequence of SEQ ID NO: 3, and the nucleic acid moleculeencoding the light chain immunoglobulin molecule represented in SEQ IDNO: 2, e.g., the nucleic acid sequence of SEQ ID NO: 1.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, (3) is expressed by a cell from adifferent species, or (4) does not occur in nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the heavychain immunoglobulin molecule represented in SEQ ID NO: 4, and the lightchain immunoglobulin molecule represented in SEQ ID NO: 2 as well asantibody molecules formed by combinations comprising the heavy chainimmunoglobulin molecules with light chain immunoglobulin molecules, suchas kappa light chain immunoglobulin molecules, and vice versa, as wellas fragments and analogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein means apolymer of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes, although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotides ofthe invention are either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes Oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. SeeDayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110(Volume 5, National Biomedical Research Foundation (1972)) andSupplement 2 to this volume, pp. 1-10. The two sequences or partsthereof are more preferably homologous if their amino acids are greaterthan or equal to 50% identical when optimally aligned using the ALIGNprogram. The term “corresponds to” is used herein to mean that apolynucleotide sequence is homologous (i.e., is identical, not strictlyevolutionarily related) to all or a portion of a referencepolynucleotide sequence, or that a polypeptide sequence is identical toa reference polypeptide sequence. In contradistinction, the term“complementary to” is used herein to mean that the complementarysequence is homologous to all or a portion of a reference polynucleotidesequence. For illustration, the nucleotide sequence “TATAC” correspondsto a reference sequence “TATAC” and is complementary to a referencesequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland? Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long' morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has specific bindingto one or more Frizzled receptors, under suitable binding conditions.Typically, polypeptide analogs comprise a conservative amino acidsubstitution (or addition or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986), Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987). Such compounds are often developed with theaid of computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—-(cis and trans), —COCH₂—,CH(OH)CH₂—, and —CH₂SO—, by methods well known in the art. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) maybe used to generate more stable peptides. In addition, constrainedpeptides comprising a consensus sequence or a substantially identicalconsensus sequence variation may be generated by methods known in theart (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example,by adding internal cysteine residues capable of forming intramoleculardisulfide bridges which cyclize the peptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis is frequently a property of antineoplastic agents.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

Anti-Frizzled Receptor Antibodies and Fragments

Monoclonal antibodies and antigen-binding fragments thereof have theability to inhibit Frizzled function and/or Frizzled activation.Inhibition is determined, for example, using the materials and methodsdescribed herein.

Exemplary antibody fragments of the invention include, for example, theFab fragments having the sequences shown in FIG. 1C and encoded by thesequences shown in FIG. 1D, where the variable light chaincomplementarity determining region 1 (CDR L1 or VL CDR1) includes theamino acid sequence SVSSA (SEQ ID NO: 392) and the variable light chaincomplementarity determining region 2 (CDR L2 or VL CDR2) includes theamino acid sequence SASSLYS (SEQ ID NO: 393). The amino acidsencompassing the complementarity determining regions (CDR) are asdefined by E. A. Kabat et al. (See Kabat, E A, et al., Sequences ofProtein of immunological interest, Fifth Edition, US Department ofHealth and Human Services, US Government Printing Office (1991)).Exemplary monoclonal antibodies of the invention include, for example,antibodies having the light chain and heavy chain sequences shown inFIGS. 1A and 1B. Exemplary monoclonal antibodies of the inventioninclude, for example, antibodies having the combination ofcomplementarity determining regions (CDRs) shown in FIG. 1C and encodedby the sequences shown in FIG. 1D, where the CDR L1 includes the aminoacid sequence SVSSA (SEQ ID NO: 392) and the CDR L2 includes the aminoacid sequence SASSLYS (SEQ ID NO: 393). Alternatively, the monoclonalantibody is an antibody that binds to the same Frizzled epitope as theseantibodies or antibody fragments.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if an anti-Frizzled receptorantibody (e.g., monoclonal antibody) has the same specificity as anantibody or antibody fragment of the invention by ascertaining whetherthe former prevents the latter from binding to collagen. If themonoclonal antibody being tested competes with the monoclonal antibodyof the invention, as shown by a decrease in binding by the monoclonalantibody of the invention, then the two monoclonal antibodies bind tothe same, or a closely related, epitope.

An alternative method for determining whether a monoclonal antibody hasthe specificity of an antibody or antibody fragment of the invention isto pre-incubate the monoclonal antibody of the invention with solubleFrizzled receptor protein, and then add the monoclonal antibody beingtested to determine if the monoclonal antibody being tested is inhibitedin its ability to bind one or more Frizzled receptors. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or functionally equivalent, epitopic specificity as the monoclonalantibody of the invention.

Screening of monoclonal antibodies and antigen-binding fragmentsthereof, can be also carried out, e.g., by measuring binding between oneor more Frizzled receptors and a Wnt protein ligand, and determiningwhether the test monoclonal antibody is able to modulate, block,inhibit, reduce, antagonize, neutralize or otherwise interfere withbinding between the Frizzled receptor(s) and the Wnt protein ligand.

Various procedures known within the art may be used for the productionof monoclonal antibodies directed against one or more Frizzledreceptors, or against derivatives, fragments, analogs homologs ororthologs thereof. (See, for example, Antibodies: A Laboratory Manual,Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., incorporated herein by reference). Fully humanantibodies are antibody molecules in which the entire sequence of boththe light chain and the heavy chain, including the CDRs, arise fromhuman genes. Such antibodies are termed “human antibodies” or “fullyhuman antibodies” herein. Human monoclonal antibodies are prepared, forexample, using the procedures described in the Examples provided below.Human monoclonal antibodies can be also prepared by using the triomatechnique; the human B-cell hybridoma technique (see Kozbor, et al.,1983 Immunol Today 4: 72); and the EBV hybridoma technique to producehuman monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Humanmonoclonal antibodies may be utilized and may be produced by using humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96)

Antibodies are purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

The antibodies and fragments of the invention are monoclonal antibodies.Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize,neutralize or otherwise interfere with Frizzled receptor activity,expression and/or Wnt signaling are generated, e.g., by immunizing ananimal with membrane bound and/or soluble Frizzled receptor, such as,for example, murine, rat or human Frizzled receptor or an immunogenicfragment, derivative or variant thereof. Alternatively, the animal isimmunized with cells transfected with a vector containing a nucleic acidmolecule encoding a Frizzled receptor is expressed and associated withthe surface of the transfected cells. Alternatively, the antibodies areobtained by screening a library that contains antibody or antigenbinding domain sequences for binding to one or more Frizzled receptors.This library is prepared, e.g., in bacteriophage as protein or peptidefusions to a bacteriophage coat protein that is expressed on the surfaceof assembled phage particles and the encoding DNA sequences containedwithin the phage particles (i.e., “phage displayed library”). Hybridomasresulting from myeloma/B cell fusions are then screened for reactivityto one or more Frizzled receptors.

Monoclonal antibodies are prepared, for example, using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of monoclonalantibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (MA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies and antigen-binding fragments thereof can bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Thehybridoma cells of the invention serve as a preferred source of suchDNA. Once isolated, the DNA can be placed into expression vectors, whichare then transfected into host cells such as simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The DNA also can be modified,for example, by substituting the coding sequence for human heavy andlight chain constant domains in place of the homologous murine sequences(see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

Human Antibodies and Humanization of Antibodies

Monoclonal antibodies and antigen-binding fragments thereof includefully human antibodies or humanized antibodies. These antibodies aresuitable for administration to humans without engendering an immuneresponse by the human against the administered immunoglobulin.

An anti-Frizzled receptor antibody or fragment is generated, forexample, using the procedures described in the Examples provided below.

In some methods, an anti-Frizzled receptor antibody or fragment isdeveloped, for example, using phage-display methods using antibodiescontaining only human sequences. Such approaches are well-known in theart, e.g., in WO92/01047 and U.S. Pat. No. 6,521,404, which are herebyincorporated by reference. In this approach, a combinatorial library ofphage carrying random pairs of light and heavy chains are screened usingnatural or recombinant source of a Frizzled receptor or fragmentsthereof. In another approach, an antibody or fragment can be produced bya process wherein at least one step of the process includes immunizing atransgenic, non-human animal with a human Frizzled receptor protein. Inthis approach, some of the endogenous heavy and/or kappa light chainloci of this xenogenic non-human animal have been disabled and areincapable of the rearrangement required to generate genes encodingimmunoglobulins in response to an antigen. In addition, at least onehuman heavy chain locus and at least one human light chain locus havebeen stably transfected into the animal. Thus, in response to anadministered antigen, the human loci rearrange to provide genes encodinghuman variable regions immunospecific for the antigen. Uponimmunization, therefore, the xenomouse produces B-cells that secretefully human immunoglobulins.

A variety of techniques are well-known in the art for producingxenogenic non-human animals. For example, see U.S. Pat. Nos. 6,075,181and 6,150,584, which is hereby incorporated by reference in itsentirety. This general strategy was demonstrated in connection withgeneration of the first XenoMouse™ strains as published in 1994. SeeGreen et al. Nature Genetics 7:13-21 (1994), which is herebyincorporated by reference in its entirety. See also, U.S. Pat. Nos.6,162,963, 6,150,584, 6, 114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2 and EuropeanPatent No., EP 0 463 151 B1 and International Patent Applications No. WO94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and related familymembers.

In an alternative approach, others have utilized a “minilocus” approachin which an exogenous Ig locus is mimicked through the inclusion ofpieces (individual genes) from the Ig locus. Thus, one or more VH genes,one or more D_(H) genes, one or more J_(H) genes, a mu constant region,and a second constant region (preferably a gamma constant region) areformed into a construct for insertion into an animal. See e.g., U.S.Pat. Nos. 5,545,806; 5,545,807; 5,591,669; 5,612,205; 5,625,825;5,625,126; 5,633,425; 5,643,763; 5,661,016; 5,721,367; 5,770,429;5,789,215; 5,789,650; 5,814,318; 5,877; 397; 5,874,299; 6,023,010; and6,255,458; and European Patent No. 0 546 073 B 1; and InternationalPatent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO97/13852, and WO 98/24884 and related family members.

Generation of human antibodies from mice in which, through microcellfusion, large pieces of chromosomes, or entire chromosomes, have beenintroduced, has also been demonstrated. See European Patent ApplicationNos. 773 288 and 843 961.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and a immune variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against one or more Frizzled receptors in order to vitiate orotherwise mitigate concerns and/or effects of HAMA or HACA response.

The production of antibodies with reduced immunogenicity is alsoaccomplished via humanization, chimerization and display techniquesusing appropriate libraries. It will be appreciated that murineantibodies or antibodies from other species can be humanized orprimatized using techniques well known in the art. See e.g., Winter andHarris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews inImmunol. 12125-168 (1992). The antibody of interest may be engineered byrecombinant DNA techniques to substitute the CH1, CH2, CH3, hingedomains, and/or the framework domain with the corresponding humansequence (See WO 92102190 and U.S. Pat. Nos. 5,530,101; 5,585,089;5,693,761; 5,693,792; 5,714,350; and 5,777,085). Also, the use of IgcDNA for construction of chimeric immunoglobulin genes is known in theart (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521(1987)). mRNA is isolated from a hybridoma or other cell producing theantibody and used to produce cDNA. The cDNA of interest may be amplifiedby the polymerase chain reaction using specific primers (U.S. Pat. Nos.4,683,195 and 4,683,202). Alternatively, a library is made and screenedto isolate the sequence of interest. The DNA sequence encoding thevariable region of the antibody is then fused to human constant regionsequences. The sequences of human constant regions genes may be found inKabat et al. (1991) Sequences of Proteins of immunological Interest,N.I.H. publication no. 91-3242. Human C region genes are readilyavailable from known clones. The choice of isotype will be guided by thedesired effecter functions, such as complement fixation, or activity inantibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1,IgG3 and IgG4. Either of the human light chain constant regions, kappaor lambda, may be used. The chimeric, humanized antibody is thenexpressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g., by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g., SV-40 earlypromoter, (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcomavirus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murineleukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)). Also, as willbe appreciated, native Ig promoters and the like may be used.

Further, human antibodies or antibodies from other species can begenerated through display type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes andPluckthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley andSmith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol.17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel etal. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol.Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A(1992), and U.S. Pat. No. 5,733,743. If display technologies areutilized to produce antibodies that are not human, such antibodies canbe humanized as described above.

Using these techniques, antibodies can be generated to Frizzledreceptor-expressing cells, soluble forms of one or more Frizzledreceptors or combination thereof, epitopes or peptides thereof, andexpression libraries thereto (See e.g., U.S. Pat. No. 5,703,057) whichcan thereafter be screened as described above for the activitiesdescribed herein.

The anti-Frizzled receptor antibodies and fragments of the invention canbe expressed by a vector containing a DNA segment encoding the singlechain antibody described above.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g. a ligand to acellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways. For example, to detect the presence ofone or more Frizzled receptors in a sample. The antibody can also beused to try to bind to and disrupt a Frizzled receptor-related activity.

Techniques can be adapted for the production of single-chain antibodiesspecific to an antigenic protein of the invention (see e.g., U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof Fab expression libraries (see e.g., Huse, et al., 1989 Science 246:1275-1281) to allow rapid and effective identification of monoclonalF_(ab) fragments with the desired specificity for a protein orderivatives, fragments, analogs or homologs thereof. Antibody fragmentsthat contain the idiotypes to a protein antigen may be produced bytechniques known in the art including, but not limited to: (i) anF_((ab′)2) fragment produced by pepsin digestion of an antibodymolecule; (ii) an Fab fragment generated by reducing the disulfidebridges of an F_((ab′)2) fragment; (iii) an F_(ab) fragment generated bythe treatment of the antibody molecule with papain and a reducing agentand (iv) Fv fragments.

The invention also includes F_(v), F_(ab), F_(ab′) and F_((ab′)2)anti-Frizzled receptor antibody fragments, single chain anti-Frizzledreceptor antibodies, bispecific anti-Frizzled receptor antibodies, andheteroconjugate anti-Frizzled receptor antibodies.

Bispecific antibodies are antibodies that have binding specificities forat least two different antigens. In the present case, one of the bindingspecificities is for a Frizzled receptor or a combination of Frizzledreceptors. The second binding target is any other antigen, andadvantageously is a cell-surface protein or receptor or receptorsubunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells which express a particularantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein and further binds tissuefactor (TF).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating diseases and disorders associated with aberrantFrizzled receptor activation and/or activity. For example, cysteineresidue(s) can be introduced into the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated can have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)).Alternatively, an antibody can be engineered that has dual Fc regionsand can thereby have enhanced complement lysis and ADCC capabilities.(See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹² Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies andantigen-binding fragments thereof. (See, for example, “ConjugateVaccines”, Contributions to Microbiology and Immunology, J. M. Cruse andR. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entirecontents of which are incorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

Preferred linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NETS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Use of Antibodies Against Frizzled Receptors

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

In one embodiment, antibodies and antigen-binding fragments thereof,which include a monoclonal antibody of the invention, may be used astherapeutic agents. Such agents will generally be employed to diagnose,prognose, monitor, treat, alleviate, and/or prevent a disease orpathology associated with aberrant Frizzled receptor activity and/oractivation in a subject. A therapeutic regimen is carried out byidentifying a subject, e.g., a human patient suffering from (or at riskof developing) a disease or disorder associated with aberrant Frizzledreceptor activity, e.g., cancer or an inflammatory disorder, usingstandard methods. An antibody preparation, preferably one having highspecificity and high affinity for its target antigen, is administered tothe subject and will generally have an effect due to its binding withthe target. Administration of the antibody may abrogate or inhibit orinterfere with a function of the target (e.g., one or more Frizzledreceptors). Administration of the antibody may abrogate or inhibit orinterfere with the binding of the target (e.g., one or more Frizzledreceptor) with an endogenous ligand (e.g., collagen) to which itnaturally binds. Administration of the antibody may modulate, block,inhibit, reduce, antagonize, neutralize, or otherwise interfere with oneor more biological activities of one or more Frizzled receptors.

Diseases or disorders related to aberrant Frizzled receptor activity,activation, expression and/or Wnt signaling include cancers andinflammatory disorders.

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between theantibody and its target antigen that, in certain cases, interferes withthe functioning of the target. The amount required to be administeredwill furthermore depend on the binding affinity of the antibody for itsspecific antigen, and will also depend on the rate at which anadministered antibody is depleted from the free volume other subject towhich it is administered. Common ranges for therapeutically effectivedosing of an antibody or antibody fragment of the invention may be, byway of nonlimiting example, from about 0.1 mg/kg body weight to about 50mg/kg body weight. Common dosing frequencies may range, for example,from twice daily to once a week.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular cancer and/orinflammatory-related disorder. Alleviation of one or more symptoms ofthe cancer and/or inflammatory-related disorder indicates that theantibody confers a clinical benefit.

Methods for the screening of antibodies that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA) and other immunologically mediated techniques known withinthe art.

In another embodiment, antibodies and fragments directed against one ormore Frizzled receptors may be used in methods known within the artrelating to the localization and/or quantitation of one or more Frizzledreceptors (e.g., for use in measuring levels of one or more Frizzledreceptors within appropriate physiological samples, for use indiagnostic methods, for use in imaging the protein, and the like). In agiven embodiment, antibodies specific to one or more Frizzled receptors,or derivative, fragment, analog or homolog thereof, that contain theantibody derived antigen binding domain, are utilized aspharmacologically active compounds (referred to hereinafter as“Therapeutics”).

In another embodiment, an antibody or fragment specific for one or moreFrizzled receptors can be used to isolate a Frizzled receptorpolypeptide, by standard techniques, such as immunoaffinity,chromatography or immunoprecipitation. Antibodies directed against oneor more Frizzled receptors (or a fragment thereof) can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure to, for example, determine the efficacy of a giventreatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In yet another embodiment, an antibody according to the invention can beused as an agent for detecting the presence of one or more Frizzledreceptors (or a protein fragment thereof) in a sample. In someembodiments, the antibody contains a detectable label. Antibodies arepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., F_(ab), scFv, or F_((ab)2)) is used. The term“labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently-labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently-labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. Included within the usage of the term“biological sample”, therefore, is blood and a fraction or component ofblood including blood serum, blood plasma, or lymph. That is, thedetection method of the invention can be used to detect an analyte mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of an analyte mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of an analyte protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations, andimmunofluorescence. In vitro techniques for detection of an analytegenomic DNA include Southern hybridizations. Procedures for conductingimmunoassays are described, for example in “ELISA: Theory and Practice:Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) HumanPress, Totowa, N.J., 1995; “Immunoassay”, E. Diamandis and T.Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and“Practice and Theory of Enzyme Immunoassays”, P. Tijssen, ElsevierScience Publishers, Amsterdam, 1985. Furthermore, in vivo techniques fordetection of an analyte protein include introducing into a subject alabeled anti-analyte protein antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

Therapeutic Administration and Formulations of Anti-Frizzled ReceptorAntibodies and Fragments

The antibodies of the invention (also referred to herein as “activecompounds”), and derivatives, fragments, analogs and homologs thereof,can be incorporated into pharmaceutical compositions suitable foradministration. Principles and considerations involved in preparing suchcompositions, as well as guidance in the choice of components areprovided, for example, in Remington's Pharmaceutical Sciences: TheScience And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al.,editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement:Concepts, Possibilities, Limitations, And Trends, Harwood AcademicPublishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery(Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

Such compositions typically comprise the antibody and a pharmaceuticallyacceptable carrier. Where antibody fragments are used, the smallestinhibitory fragment that specifically binds to the binding domain of thetarget protein is preferred. For example, based upon the variable-regionsequences of an antibody, peptide molecules can be designed that retainthe ability to bind the target protein sequence. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology.(See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893(1993)).

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a sustained/controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

For example, the active ingredients can be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

The materials can also be obtained commercially from Alza Corporationand Nova Pharmaceuticals, Inc. Liposomal suspensions (includingliposomes targeted to infected cells with monoclonal antibodies to viralantigens) and can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The formulation can also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

In one embodiment, the active compounds are administered in combinationtherapy, i.e., combined with other agents, e.g., therapeutic agents,that are useful for treating pathological conditions or disorders, suchas various forms of cancer and inflammatory diseases. The term “incombination” in this context means that the agents are givensubstantially contemporaneously, either simultaneously or sequentially.If given sequentially, at the onset of administration of the secondcompound, the first of the two compounds is preferably still detectableat effective concentrations at the site of treatment.

For example, the combination therapy can include one or more antibodiesand antigen-binding fragments thereof coformulated with, and/orcoadministered with, one or more additional therapeutic agents, e.g.,one or more cytokine and growth factor inhibitors, immunosuppressants,anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors,antineoplastic agents, and/or cytotoxic or cytostatic agents, asdescribed in more detail below. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

Preferred therapeutic agents used in combination with an antibody of theinvention are those agents that interfere at different stages in aninflammatory response. In one embodiment, one or more antibodiesdescribed herein may be coformulated with, and/or coadministered with,one or more additional agents such as other cytokine or growth factorantagonists (e.g., soluble receptors, peptide inhibitors, smallmolecules, ligand fusions); or antibodies or antigen binding fragmentsthereof that bind to other targets (e.g., antibodies that bind to othercytokines or growth factors, their receptors, or other cell surfacemolecules); and anti-inflammatory cytokines or agonists thereof.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto Frizzled receptors, the design of other therapeutic modalities beyondantibody moieties is facilitated. Such modalities include, withoutlimitation, advanced antibody therapeutics, such as bispecificantibodies, immunotoxins, and radiolabeled therapeutics, generation ofpeptide therapeutics, gene therapies, particularly intrabodies,antisense therapeutics, and small molecules.

For example, in connection with bispecific antibodies, bispecificantibodies can be generated that comprise (i) two antibodies-one with aspecificity to one or more Frizzled receptors and another to a secondmolecule that are conjugated together, (ii) a single antibody that hasone chain specific to one or more Frizzled receptors and a second chainspecific to a second molecule, or (iii) a single chain antibody that hasspecificity to one or more Frizzled receptors and a second molecule.Such bispecific antibodies are generated using techniques that are wellknown for example, in connection with (i) and (ii) See e.g., Fanger etal. Immunol Methods 4:72-81 (1994) and Wright et al. Crit, Reviews inImmunol. 12125-168 (1992), and in connection with (iii) See e.g.,Traunecker et al. Int. J. Cancer (Suppl.) 7:51-52 (1992).

In connection with immunotoxins, antibodies can be modified to act asimmunotoxins utilizing techniques that are well known in the art. Seee.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902. Each of immunotoxins and radiolabeled molecules would belikely to kill cells expressing one or more Frizzled receptors.

In connection with the generation of therapeutic peptides, through theutilization of structural information related to one or more Frizzledreceptors and antibodies thereto, such as the antibodies andantigen-binding fragments thereof or screening of peptide libraries,therapeutic peptides can be generated that are directed against one ormore Frizzled receptors. Design and screening of peptide therapeutics isdiscussed in connection with Houghten et al. Biotechniques 13:412-421(1992), Houghten PNAS USA 82:5131-5135 (1985), Pinalla et al.Biotechniques 13:901-905 (1992), Blake and Litzi-Davis BioConjugateChem. 3:510-513 (1992). Immunotoxins and radiolabeled molecules can alsobe prepared, and in a similar manner, in connection with peptidicmoieties as discussed above in connection with antibodies. Assuming thatthe Frizzled receptor molecule (or a form, such as a splice variant oralternate form) is functionally active in a disease process, it willalso be possible to design gene and antisense therapeutics theretothrough conventional techniques. Such modalities can be utilized formodulating the function of one or more Frizzled receptors. In connectiontherewith the antibodies of the present invention facilitate design anduse of functional assays related thereto. A design and strategy forantisense therapeutics is discussed in detail in International PatentApplication No. WO 94/29444. Design and strategies for gene therapy arewell known. However, in particular, the use of gene therapeutictechniques involving intrabodies could prove to be particularlyadvantageous. See e.g., Chen et al. Human Gene Therapy 5:595-601 (1994)and Marasco Gene Therapy 4:11-15 (1997). General design of andconsiderations related to gene therapeutics is also discussed inInternational Patent Application No. WO 97/38137.

Knowledge gleaned from the structure of one or more targeted Frizzledreceptor molecules and its/their interactions with other molecules inaccordance with the present invention, such as the antibodies andantigen-binding fragments thereof, and others can be utilized torationally design additional therapeutic modalities. In this regard,rational drug design techniques such as X-ray crystallography,computer-aided (or assisted) molecular modeling (CAMM), quantitative orqualitative structure-activity relationship (QSAR), and similartechnologies can be utilized to focus drug discovery efforts. Rationaldesign allows prediction of protein or synthetic structures which caninteract with the molecule or specific forms thereof which can be usedto modify or modulate the activity of one or more Frizzled receptors.Such structures can be synthesized chemically or expressed in biologicalsystems. This approach has been reviewed in Capsey et al. GeneticallyEngineered Human Therapeutic Drugs (Stockton Press, N.Y. (1988)).Further, combinatorial libraries can be designed and synthesized andused in screening programs, such as high throughput screening efforts.

Screening Methods

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules or otherdrugs) that modulate or otherwise interfere with the binding of one ormore Frizzled receptors to a Wnt protein ligand and/or with Frizzledactivation, or candidate or test compounds or agents that modulate orotherwise interfere with Wnt signaling function. Also provided aremethods of identifying compounds useful to treat disorders associatedwith aberrant Frizzled receptor activity, activation, expression and/orphosphorylation. The invention also includes compounds identified in thescreening assays described herein.

In one embodiment, the invention provides assays for screening candidateor test compounds which modulate a function of one or more Frizzledreceptors. The test compounds of the invention can be obtained using anyof the numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the “one-bead one-compound” library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds. (See, e.g., Lam, 1997. AnticancerDrug Design 12: 145).

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (see e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (see Lam, 1991. Nature354: 82-84), on chips (see Fodor, 1993. Nature 364: 555-556), bacteria(see U.S. Pat. No. 5,223,409), spores (see U.S. Pat. No. 5,233,409),plasmids (see Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (see Scott and Smith, 1990. Science 249: 386-390;Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl.Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409.).

In one embodiment, a candidate compound is introduced to anantibody-antigen complex and determining whether the candidate compounddisrupts the antibody-antigen complex, wherein a disruption of thiscomplex indicates that the candidate compound modulates a function ofone or more Frizzled receptor and/or the interaction between one or moreFrizzled receptors and a Wnt protein ligand.

Determining the ability of the test compound to interfere with ordisrupt the antibody-antigen complex can be accomplished, for example,by coupling the test compound with a radioisotope or enzymatic labelsuch that binding of the test compound to the antigen orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the assay comprises contacting an antibody-antigencomplex with a test compound, and determining the ability of the testcompound to interact with the antigen or otherwise disrupt the existingantibody-antigen complex. In this embodiment, determining the ability ofthe test compound to interact with the antigen and/or disrupt theantibody-antigen complex comprises determining the ability of the testcompound to preferentially bind to the antigen or a biologically-activeportion thereof, as compared to the antibody.

In another embodiment, the assay comprises contacting anantibody-antigen complex with a test compound and determining theability of the test compound to modulate the antibody-antigen complex.Determining the ability of the test compound to modulate theantibody-antigen complex can be accomplished, for example, bydetermining the ability of the antigen to bind to or interact with theantibody, in the presence of the test compound.

The screening methods disclosed herein may be performed as a cell-basedassay or as a cell-free assay. The cell-free assays of the invention areamenable to use of either the soluble form or the membrane-bound form ofone or more Frizzled receptors, and fragments thereof. In the case ofcell-free assays comprising the membrane-bound forms of one or moreFrizzled receptors, it may be desirable to utilize a solubilizing agentsuch that the membrane-bound form of the proteins are maintained insolution. Examples of such solubilizing agents include non-ionicdetergents such as n-octylglucoside, n-dodecylglucoside,n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)n,N-dodecyl--N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminio1-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment, it may be desirable to immobilize eitherthe antibody or the antigen to facilitate separation of complexed fromuncomplexed forms of one or both following introduction of the candidatecompound, as well as to accommodate automation of the assay. Observationof the antibody-antigen complex in the presence and absence of acandidate compound can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-antibodyfusion proteins or GST-antigen fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound, and the mixture is incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly.Alternatively, the complexes can be dissociated from the matrix, and thelevel of antibody-antigen complex formation can be determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theantibody (e.g. the anti-Frizzled receptor antibodies and fragments ofthe invention) or the antigen (e.g. one or more Frizzled receptors) canbe immobilized utilizing conjugation of biotin and streptavidin.Biotinylated antibody or antigen molecules can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques well-known withinthe art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, other antibodies reactive with the antibody orantigen of interest, but which do not interfere with the formation ofthe antibody-antigen complex of interest, can be derivatized to thewells of the plate, and unbound antibody or antigen trapped in the wellsby antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using such other antibodiesreactive with the antibody or antigen.

The invention further pertains to novel agents identified by any of theaforementioned screening assays and uses thereof for treatments asdescribed herein.

Diagnostic and Prophylactic Formulations

The anti-Frizzled antibodies and fragments of the invention are used indiagnostic and prophylactic formulations. In one embodiment, ananti-Frizzled antibody or fragment of the invention is administered topatients that are at risk of developing one or more of theaforementioned diseases, such as for example, without limitation, cancerand/or inflammatory disorders. A patient's or organ's predisposition toone or more of these diseases can be determined using genotypic,serological or biochemical markers.

Antibodies and antigen-binding fragments thereof are also useful in thedetection of one or more Frizzled receptors in patient samples andaccordingly are useful as diagnostics. For example, the anti-Frizzledreceptor antibodies and fragments of the invention are used in in vitroassays, e.g., ELISA, to detect Frizzled receptor levels in a patientsample.

In one embodiment, an anti-Frizzled receptor antibody or fragment of theinvention is immobilized on a solid support (e.g., the well(s) of amicrotiter plate). The immobilized antibody serves as a capture antibodyfor any Frizzled receptor that may be present in a test sample. Prior tocontacting the immobilized antibody with a patient sample, the solidsupport is rinsed and treated with a blocking agent such as milk proteinor albumin to prevent nonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of Frizzled receptor antigen in the test sample isdetermined by comparison with a standard curve developed from thestandard samples.

It will be appreciated that based on the results obtained using theanti-Frizzled receptor antibodies and fragments of the invention in anin vitro diagnostic assay, it is possible to stage a disease (e.g., aclinical indication associated with cancer or inflammatory disorder) ina subject based on expression levels of the Frizzled receptor antigen.For a given disease, samples of blood are taken from subjects diagnosedas being at various stages in the progression of the disease, and/or atvarious points in the therapeutic treatment of the disease. Using apopulation of samples that provides statistically significant resultsfor each stage of progression or therapy, a range of concentrations ofthe antigen that may be considered characteristic of each stage isdesignated.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1: Materials and Methods

Phage display selection: Phage display selections were performed onrecombinant human FZD7 extracellular domains (R&D Systems) according tothe procedure described by Sachdev S. Sidhu and Frederic A. Fellouse.“Synthetic therapeutic antibodies.” Nat Chem Biol. 2(12)(2006):682-8.

Fab expression and purification: IPTG-inducible expression plasmids wereused for the production of Fabs in BL21 bacterial cells. Briefly, 25 mlovernight bacterial cultures are back diluted in 1 L 2YT media. At anO.D.=1 cells were induced with a final concentration of 1 mM IPTG. After4 hours of induction the cells were collected by centrifugation and thepellets were frozen at −20° C. The next day pellets were resuspended in40 ml lysis buffer (1% Triton X-100, 2 mM MgCl₂, 0.2 mM PMSF, 0.1% (w/v)lysozyme and 1 ul benzonase (2.5 U/ml) prior to lysis at 4° C. for 1hour. The Fab containing supernatant was separated from cell debris bycentrifugation at 11000 RPM for 45 min. Fabs were captured at 4° C. for1 hour with Sepharose A beads, using 1 ml bead volume per 40 ml Fabsolution. The beads were collected in 25 ml columns (Biorad), followedby 3×10 ml PBS washes. Fabs were eluted with 8 ml of elution buffer (50mM NaH₂PO₄, 100 mM H₃PO₄, 140 mM NaCl, pH 2.8) and neutralized withTris-HCl pH 8.0 at a final concentration of 200 mM. PBS buffer exchangewas carried out using standard dialysis.

IgG expression and purification: IgG was expressed in 293 F cells andpurified from the condition media according to conventional methodology.Briefly, equal amounts of DNA constructs encoding light chain or heavychain were co-transfected into 293 F cells. The cell culture conditionmedia were harvested 120-145 hours post transfection. Appropriate amountof Protein A beads were added to the condition media and mixed at roomtemperature for 1-2 hours. The supernatant was removed and the beadswere washed 3 times with PBS and the IgG was then eluted with elutionbuffer (0.1 M Glycine-HCl, pH 2.7) and neutralized with neutralizationbuffer (1 M Tris-HCl, pH 9.0). The purified IgG was then dialysed andbuffer exchanged into PBS.

ELISA assays: Binding of the Fabs were assessed by immobilizing 100 nMFZD7 antigen and Fc control protein for 1 hour at RT, followed by an 1hour blocking step in 60 ul of PBS-BSA solution (for details see SachdevS. Sidhu and Frederic A. Fellouse. “Synthetic therapeutic antibodies.”Nat Chem Biol. 2(12)(2006):682-8). After blocking 100 nM Fab was addedto each well, incubated for 30 min, washed 6 times with PBS-Tween bufferand detected with anti-FLAG-HRP (1:5000 dilution).

Affinity measurement using Surface Plasmon Resonance (SPR): For kineticanalysis, Fab proteins were purified from Escherichia coli. Bindingkinetics were determined by surface plasmon resonance using a PROTEON(BioRAD) with antigen immobilized on GLC chips at a density sufficientto produce ˜100 response units when saturated with Fab. Serial dilutionsof Fab proteins were injected at 100 ul minute for 1 minute. PBS wasthen injected at 100 ul/minute for 10 minutes to observe dissociation.Binding responses were corrected by subtraction of responses on a blankflow cell. For kinetic analysis, a 1:1 Langmuir model of global fittingsof k_(on), and k_(off) was used. The K_(d) values were determined fromthe ratios of k_(on), and k_(off).

Epitope binning: Briefly, 100 nM immobilized antigen was pre-incubatedwith 500 nM purified Fab for 1 hour. Freshly prepared phage, carryingthe anti-FZD7 Fab molecules, were added to the antigen-Fab complexcontaining wells. Fabs that block overlapping phage-Fab-antigeninteractions were grouped together. For details see Sachdev S. Sidhu andFrederic A. Fellouse. “Synthetic therapeutic antibodies.” Nat Chem Biol.2(12) (2006):682-8.

Cell surface binding by flow cytometry: Cells are collected by EDTAsolution and wash with PBS once, aliquot to 0.2-1×10⁶ cells per sample;the cells were incubated with tested antibody (1 μg ab per sample in 100ul PBS-0.5% BSA) for 60 min on ice; Cells were then washed with 1 mlPBS-0.1% BSA twice and incubated with 100 ul Goat anti-Human Fab-488diluted in PBS-0.1% BSA for 1530 min at RT, in the dark. Cells were thenwashed with 1 ml PBS-0.1% BSA twice before re-suspended in filtered0.2-0.5 ml 4% Paraformaldehyde. The cell samples are subjected to FACSanalysis immediately or, to be kept at 4° C. for later analysis.Secondary Ab used: Alexa Fluor® 488 goat anti-human Fab (Jackson#109-546-097) 1:1000 EDTA Solution (for detach adherent cells): 0.15 gdisodium EDTA (1 mM), 4.0 g NaCl, 0.28 g sodium bicarbonate, 0.5 gdextrose (D-glucose), 0.2 g KCl, Dissolved in 500 ml double distilledwater.

TopFlash reporter assay: Lentivirus containing the TopFlashβ-catenin-dependent luciferase reporter (firefly luciferase) and Renillaluciferase were used to establish stable MDA-MB-231 and HEK293TWnt-reporter lines. Twenty-four hours prior treatments, cells wereseeded on 24-well plate at 50% confluency in a final volume of 500 ul.The following day, half of the media was replaced with 250 μl of Wnt3aor control conditioned media from L cells and Fabs or IgGs were added atthe desired concentration. The cells were then assayed 15 hours afterstimulation in accordance with the dual luciferase protocol (Promega)using an Envision multilabel plate reader (Perkin-Elmer).

Immunofluorescence staining: Lentivirus containing the CRD of FZDreceptors fused to a myc tag and a GPI anchor sequence were used toestablish stable CHO cells. CHO stable cell lines or MDA-MB-231 cellswere seeded on coverslips and incubated 1 hour at 4° C. in DMEM, 10%FBS, 1% Pen/Strep in presence of 200 nM of Fabs or IgG or 1:200 dilutionof anti-cMyc (SantaCruz). Cells were fixed for 15 minutes in 3.7%paraformaldehyde and block with 10% normal donkey serum (NDS) for 30minutes and incubated with secondary anti-Fab 488 Alexa fluor (Jackson)or anti-rabbit 488 Alexa Fluor (Molecular Probe) with 0.1% NDS for 60minutes at room temperature. Coverslips were mounted with Vectashield(Vector Laboratories Inc) containing DAPI and imaged with a NikonEclipse 80i or a LSM 700 (Zeiss).

The Sulforhodamine B (SRB) assay to measure cell growth: Cancer cellswere plated in a 96 well plate with the following densities: ASPC1 at1800/well, HPAFII at 1500/well, CAPAN2 at 3000/well and IMIMPC2 at600/well. Cells were cultured overnight, followed by the addition ofanti-FZD7 IgGs to the cells in a total volume of 100 μl of culturemedium and kept in the incubator for 5 days. The cells were subsequentlyfixed in situ by gently aspirating off the culture medium, followed bythe addition of 50 μl of ice cold 10% TAC (Tri-chloroacetic Acid) toeach well and incubated at 4° C. for 30-60 min.

The plates were washed with tap water five times and allowed to air dryfor 5 minutes. Subsequently, 50 μl of 0.4% Sulforhodamine B solution in1% acetic acid were added to each well and incubated for 30 min at roomtemperature for staining. Following staining, plates were washed fourtimes with freshly-made 1% acetic acid to remove any unbound dye andthen allowed to air dry for 5 mins. The stain was solubilized with 100μl of 10 mM Tris (PH 10.5) per well. The plate was then placed on anorbital rotator for 5 minutes before Absorbance was read at 570 nm.

Cell culture: Human pancreatic adenocarcinoma cell lines, AsPC-1, CAPAN2and HPAFII were purchased from the American Type Culture Collection(Manassas, Va., USA). The AsPC-1 cells were maintained in RPMI1640medium (Life Technologies, Grand Island, N.Y., USA. Cat #: A10491-01)supplemented with 10% FCS (Life Technologies, Grand Island, N.Y., USA.Cat #: 12483-020), 100 U/ml of penicillin and 100 U/ml streptomycin(Life Technologies, Grand Island, N.Y., USA. Cat #: 15140-122) at 37° C.in a humidified atmosphere containing 5% CO₂.

The CAPAN2 cells were maintained in McCoy's 5a medium (LifeTechnologies, Grand Island, N.Y., USA. Cat #: 16600) supplemented with10% FCS (Life Technologies, Grand Island, N.Y., USA. Cat #: 12483-020),100 U/ml of penicillin and 100 U/ml streptomycin (Life Technologies,Grand Island, N.Y., USA. Cat #: 15140-122) at 37° C. in a humidifiedatmosphere containing 5% CO₂.

The HPAFII cells were maintained in EMEM medium (Wisent Inc., St-Bruno,QC, Canada. Cat #: 320-026-CL) supplemented with 10% FCS (LifeTechnologies, Grand Island, N.Y., USA. Cat #: 12483-020), 100 U/ml ofpenicillin and 100 U/ml streptomycin (Life Technologies, Grand Island,N.Y., USA. Cat #: 15140-122) at 37° C. in a humidified atmospherecontaining 5% CO₂.

The cells were confirmed to be free from mycoplasma by periodic testingwith a MycoAlert® Mycoplasma Detection Kit (Lonza, Rockland, Me., USA.Cat #: LT07-318).

Test Antibodies (for in vivo experiments): Five recombinantanti-Frizzled 7 antibodies (FZD7 Abs) were tested in these experiments.These antibodies were A1 (mAb #105), E4 (mAb #107), H10 (mAb #111), H1(mAb #112) and G2 (mAb #140). Each antibody was diluted from its stockconcentration with vehicle buffer to the dose concentration (1.33mg/ml—equal to 20 mg/kg at a dose volume of 15 ml/kg). Vehicle bufferwas DPBS (without calcium and magnesium, Life Technologies, GrandIsland, N.Y., USA. Cat #: 14190-144) containing 5% of d-(+)-TrehaloseDihydrate (Bioshop, Burlington, ON, Canada. Cat #: TRE222).

For the dose response study, two anti-Frizzled antibodies (FZD7 Abs), Ab111 and Ab 112, were tested with 3 different dose levels (50, 20 and 5mg/kg) in these experiments. Each antibody was diluted from its stockconcentration with vehicle buffer to the dose concentrations (3.33, 1.33and 0.33 mg/ml—equal to 50, 20 and 5 mg/kg respectively at a dose volumeof 15 ml/kg). Vehicle buffer was DPBS (without calcium and magnesium,Life Technologies, Grand Island, N.Y., USA. Cat #: 14190-144) containing5% of d-(+)-Trehalose Dihydrate (Bioshop, Burlington, ON, Canada. Cat #:TRE222).

For testing in CAPAN2 and HPAFII xenograft models, anti-Frizzled 7antibody (FZD7 Ab), Ab111, was tested with 2 different dose levels (20and 5 mg/kg) in these experiments. The antibody was diluted from itsstock concentration with vehicle buffer to the dose concentrations (1.33and 0.33 mg/ml—equal to 20 and 5 mg/kg respectively at a dose volume of15 ml/kg). Vehicle buffer was DPBS (without calcium and magnesium, LifeTechnologies, Grand Island, N.Y., USA. Cat #: 14190-144).

Example 2: Selection, Generation and Characterization of Fabs AgainstFrizzled Receptors

A synthetic Fab phage display library referred to as “Library F” wasused to generate antibodies against Frizzled receptors. Usingrecombinant Frizzled receptor 7 (FZD7) cysteine-rich domain (CRD) fusedto the Fc region of human IgG as the antigen, four rounds of sequentialselection with Library F were performed, and multiple phage Fab clonesthat specifically bind to the FZD7-CRD-Fc but not Fc control wereidentified (see Materials and Methods).

Library F is a single framework human Fab library constructed similarlyto previously described libraries. (See e.g., Fellouse F A, Pal G,“Methods for the Construction of Phage-Displayed Libraries” in PhageDisplay in Biotechnology and Drug Discovery. Boca Raton: CRC Press(2005); and Fellouse F A et al. “High-throughput generation of syntheticantibodies from highly functional minimalist phage-displayed libraries.”J Mol Biol 373 (2007): 924-940).

Library F is an Fab-phage library that was constructed by introducingdegenerate codons into positions in CDR-H1, CDR-H2, CDR-H3 and CDR-L3 ofa single human Fab framework. The loop length of the CDR-L3 and/orCDR-H3 in Library F can vary as shown in the table below. In thislibrary, CDR-L1 includes the amino acid sequence SVSSA (SEQ ID NO: 392)and the CDR L2 includes the amino acid sequence SASSLYS (SEQ ID NO:393). The library has a total diversity of 3×10¹⁰ unique clones, and thedetails of the library design are shown in Table 1 below, where thebolded positions in the CDR-L3 and CDR-H3 regions represent positionsthat were replaced by random loops of all possible varying lengths, asindicated.

TABLE 1 CDR Sequences of Library F clones CDR L3 (SEQ ID NO: 394)Position 91 92 93 94 95 96 Z Z Z Z PL IL Z = 25% Y, 20% S, 20% G, 10% Aand 5% each of F, W, H, P, V Loop Length (8-12 aa) CDR H1 (SEQ ID NO:395) Position 29 30 31 32 33 34 IL YS YS YS YS IM CDR H2 (SEQ ID NO:396) Position 50 51 52 52a 53 54 55 56 57 58 YS I YS PS YS YS GS YS T YSCDR H3 (SEQ ID NO: 397) Position 94 95 96 97 98 99 100 100a 100b 100c101 R Z Z Z Z Z Z Z AG FILM D Z = 25% Y, 20% S, 20% G, 10% A and 5% eachof F, W, H, P, V Loop Length (5-22 aa)

Sequencing and sequence analysis of these clones led to identificationof 61 Fab clones with unique sequences (FIG. 1). These clones were thencloned into IPTG-inducible E. coli expression vector, and Fab proteinswere expressed and purified (FIG. 2A). It was confirmed that thesepurified anti-FZD7 Fabs bind to the FZD7-CRD-Fc, but not to the Fccontrol (FIG. 2B).

The binding of the purified Fabs to the FZD7 expressed on cells wastested. To see if these Fabs recognize the FZD7 receptor expressed oncells, a flow cytometry analysis was performed. The Fabs, except FabsA2, A12, C3, E3 and E7, bound to cells that are known to express FZD7 aswell as to the same cells that have been engineered to over-express FZD7(FIG. 3). The Fabs that showed good binding in the flow cytometry assaywere selected and purified for further analysis.

The binding specificity of the Fab phage clones was tested. To determineif these Fab clones also bound to other Frizzled receptors, phagebinding ELISA assay was carried out using purified FZD ECD-Fc fusionproteins. This analysis demonstrated that, in addition to FZD7, the Fabphage clones also bound to other Frizzled receptors (FIG. 4A). Thebinding specificities of these Fabs were further determined byimmunofluorescence staining using cells overexpressing individual FZDECDs. Fabs A1, D10, E4, G2, H3 and H10 shared the same bindingspecificity profile, which is different from those of Fab E8, G6 or H1(FIG. 4B).

Next, epitope binning of the anti-FZD Fabs was performed. To learn ifthe anti-FZD7 Fabs share binding sites on the antigen FZD7, competitiveELISA assays were performed to determine if purified Fabs would competeswith Fab phage clones for binding to the antigen (FZD7 CRD-Fc). Theseexperiments showed that Fab effectively competed with its owncorresponding phage clone (FIG. 5). In addition, Fabs G6, H1, A1 and E4effectively blocked binding of all other Fab phage clones, while Fab D10only effectively blocked the binding of Fab phage clone E8 (FIG. 5),suggesting these Fabs share overlapping binding sites on FZD7.

Next, the affinity of the anti-FZD7 Fabs was determined by SurfacePlasmon Resonance (SPR). SPR was used to determine the binding affinityof the FZD7 Fabs to FZDs. FIG. 6, which summarizes the binding affinityobtained by SPR, demonstrates that the affinity values of these Fabs forFZD7 ranged from 0.4-9 nM. Binding affinity for a subset of additionalFZDs was also obtained (FIG. 7).

The effect of the anti-FZD Fabs on Wnt ligand binding and ligand-inducedtranscription was analyzed. An ELISA assay was used to directlydetermine if the Fabs were able to block Wnt ligand binding to FZD7. Asshown in FIG. 8, Fabs A1, D10, E4, G2, H3, H10, G6 or H1 effectivelyinhibited ligand Wnt 5a binding. But Fab E8 did not block the binding ofWnt5a to FZD7. Further, in a transcriptional reporter assay, Fabs A1,G2, H1, H3, H10 and E4 inhibited Wnt 3a-induced transcriptional activityby more than 70% in comparison to the PBS control (FIG. 9).

The effect of anti-FZD mAb (IgGs) on ligand Wnt3a-inducedtranscriptional activity was also analyzed. The anti-FZD Fabs wereconverted into full-length antibodies as IgG₁, and the mAbs wereexpressed and purified from mammalian cells. The purified antibodieswere subsequently used for biochemical and biological characterizationstudies.

First the binding affinities of these mAbs to FZD7 were determined usingSPR method (see Materials and Methods). As shown in FIG. 7, K_(D) valueswere estimated ranging from 0.2-1.5 nM. Second, the antibodies wereexamined in a flow cytometry analysis to determine if these mAbs bind toFZD7 expressed on cells. The antibodies were shown to be bind to theFZD7-overexpressing CHO cells better than CHO cells, indicating theantibodies recognized FZD7 expressed on cells (FIG. 10).

The anti-FZD7 antibodies were also tested in a ligand (Wnt3a)-dependenttranscriptional reporter assay to see if they blocked signaltransduction mediated by Wnt ligand and FZD7. As shown in FIG. 11, IgGsA1, G2, H1, H3, E4, D10, H10 and E8 inhibited Wnt 3a-inducedtranscriptional activity in a dose-dependent manner. The IC₅₀ valueswere estimated ranging from 2 nM to 125 nM (FIG. 11).

Example 3: In vitro Assessment of Tumor Response FollowingAdministration of Anti-FZD7 mAbs

Human pancreatic cancer cell lines, ASPC1, HPAFII, CAPAN2 and IMMIPC2were used to assess the response of cancer cell proliferation followingtreatment with anti-FZD7 mAbs. For these studies the pancreatic celllines were plated in 96 well plates at the following densities: ASPC1cells were plated at 1800 cells/well, HPAFII cells were plated at 1500cells/well, CAPAN2 cells were plated at 3000 cells/well, and IMMIPC2cells were plated at 600 cells per well. Following an overnight cultureperiod, anti-FZD7 mAbs (H10 (mAb #111), G2 (mAb #140), A1 (mAb #105), E4(mAb #107), or 18R5) or as a negative control, human gamma globulin,were added to the cells for 5 days.

A dose dependent response in cancer cell proliferation was also shownfor the cancer cell lines HPAFII, IMMPC2, PANC08.13, and ASPC1 followingadministration of anti-FZD7 mAbs. Following a 5 day treatment of either10 μg/ml or 50 μg/ml of mAb H10 or G2, there was a dose dependentresponse in cell proliferation for the HPAFII, IMMPC2 and PANC08.13cells. (See FIG. 12A-12D). Anti-FZD7 antibodies A1 (mAb #105), E4 (mAb#107), H10 (mAb #111), H1 (mAb #112), G2 (mAb #140), and 18R5 were alsoshown to have a dose dependent response in the reduction of cellproliferation of Capan-2 cells. (See FIG. 12E).

For the in vitro experiments using ASPC-1 cells the concentrations ofthe antibodies added to the culture medium included 1 μg/ml, 2 μg/ml, 5μg/ml, 10 μg/ml, or 50 μg/ml. Dose-dependent inhibition of cellproliferation was shown for mAbs A1, E4, and H1. (See FIGS. 13A and13B).

Example 4: In Vivo Assessment of Tumor Response Following Administrationof Anti-FZD7 mAbs

Five to six week old female C.B-17 SCID mice (Taconic Farms, Germantown,N.Y., USA) received transplants of one of three pancreatic cell lines,AsPC1, CAPAN2 or HPAFII, followed by administration of anti-FZD7 mAbs toassess response to mAb treatment. Below are detailed descriptions of thein vivo work and results.

AsPC1 Transplants: C.B-17 SCID mice were injected in the right flankwith 3 million AsPC1 cells in 100 μl of DPBS, followed by a 6 day restperiod to allow the formation of an average tumor volume of 184.9 mm³.There were a total of 6 experimental groups, with each group containing9 mice. The anti-FZD7 antibodies, A1 (mAb #105), E4 (mAb #107), H10 (mAb#111), H01 (mAb #112), G2 (mAb #140), or a vehicle control (100 μlDPBS), were administered via IP twice per week, for a total of 5 weeks,at a concentration of 20 mg/kg. All anti-FZD7 mAbs resulted in a markeddecrease in tumor volume after the 5 week dosing regimen, ranging from a25.6% reduction (following administration of mAb A1), to a 50.8%reduction (following administration of mAb H10). (See FIG. 14).

Dose dependent responses in the AsPC1 mouse xenografts were alsoassessed with mAb111 (also referred to herein as H10) and mAb112 (alsoreferred to herein as H1). Mouse xenograft recipients began treatmentfollowing 6 days of tumor growth (average volume=193.6 mm³) with dosagesof 5 mg/kg, 20 mg/kg, 50 mg/kg, or vehicle only (DPBS) control, twiceper week for a total of 4 weeks. For these experiments there were atotal of 8 groups, wherein each group contained 9 animals, except forthe DPBS control group which contained 8 animals. There was a dosedependent decrease in tumor volume size following treatment, rangingfrom a 36.3% reduction in tumor volume with the 5 mg/kg dose, to a 65.8%reduction in tumor volume with the 50 mg/kg dose, followingadministration of mAb111; following treatment with mAb112, there was areduction in tumor volume size ranging from 3 5.4% (5 mg/kg dose) to44.7% (50 mg/kg). (See FIG. 15).

CAPAN2 Transplants: C.B-17 SCID mice were injected in the right flankwith 4 million AsPC1 cells, followed by a 6 day rest period to allow theformation of an average tumor volume of 203.6 mm³. For theseexperiments, anti-FZD7 mAb, mAb111, was administered via IP in dosagesof either 5 mg/kg or 20 mg/kg, or as a control, vehicle only (DPBS). Thedosage regimen was administered twice per week for a total of 5 weeks.The mice were distributed randomly into 3 groups, with each groupcontaining 10 mice. Administration of mAb111 resulted in reduction ofthe tumor volume by 52% following 5 weeks of treatment. (See FIG. 16).

HPAFII Transplants: C.B-17 SCID mice were injected in the right flankwith 3 million AsPC1 cells, followed by a 7 day rest period to allow theformation of an average tumor volume of 191.5 mm³. For theseexperiments, anti-FZD7 mAb, mAb111, was administered via IP in dosagesof either 5 mg/kg or 20 mg/kg, or as a control, vehicle only (DPBS). Thedosage regimen was administered twice per week for a total of 4.5 weeks.The mice were distributed randomly into 3 groups, with each groupcontaining 10 mice. Administration of mAb111 resulted in reduction ofthe tumor volume by 94.1% following 5 weeks of treatment. (See FIG. 17).

What is claimed is:
 1. An isolated antibody or antigen-binding fragment thereof that binds to one or more Frizzled receptors and prevents the one or more Frizzled receptors from binding to a Wnt protein ligand, wherein the antibody or antigen-binding fragment thereof comprises: a) a variable light chain complementarity determining region 1 (CDRL1) comprising an amino acid sequence of SEQ ID NO: 392; b) a variable light chain complementarity determining region 2 (CDRL2) comprising an amino acid sequence of SEQ ID NO: 393; c) a variable light chain complementarity determining region 3 (CDRL3) comprising an amino acid sequence of SEQ ID NO: 132; d) a variable heavy chain complementarity determining region (CDRH1) comprising an amino acid sequence of SEQ ID NO: 133; e) a variable heavy chain complementarity determining region (CDRH2) comprising an amino acid sequence of SEQ ID NO: 134; and f) a variable heavy chain complementarity determining region (CDRH3) comprising an amino acid sequence of SEQ ID NO:
 135. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is an IgG isotype.
 3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is an IgG1 isotype.
 4. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of claim 1 and a carrier.
 5. A method of alleviating a symptom of a disease or disorder associated with aberrant Frizzled receptor expression or activity in a subject, the method comprising administering the antibody or antigen-binding fragment thereof of claim 1 to a subject in need thereof in an amount sufficient to alleviate the symptom of the disease or disorder associated with aberrant Frizzled receptor expression or activity.
 6. The method of claim 5, wherein the subject is a human.
 7. The method of claim 5, wherein the disease or disorder associated with aberrant Frizzled receptor expression or activity is cancer.
 8. The method of claim 7, wherein the cancer is selected from breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, gastrointestinal (GI) cancer, neuroblastoma, renal cancer, prostate cancer, melanoma, leukemia or Wilm's tumor. 